Harold L. Atwood

Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na∶K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.

The GTPase dMiro is required for axonal transport of mitochondria to drosophila synapses

We have identified EMS-induced mutations in Drosophila Miro (dMiro), an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in dmiro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca2+ buffering is only impaired during prolonged stimulation. Neuronal, but not muscular, expression of dMiro in dmiro mutants restored viability, transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, our findings suggest that dMiro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals.

Organization and synaptic physiology of crustacean neuromuscular systems.

5. Transmitter substances 5.1. Inhibitory transmitter substance 5.1.1. Evidence for GABA as a transmitter 5.1.2. Effects of GABA on muscle fiber membrane receptors 5.1.3. The GABA receptors of excitatory nerve terminals 5.1.4. Uptake of GABA 5.2. Excitatory transmitter substances 5.2.1. Acetylcholine 5.2.2. Glutamate 5,2.3. Glutamate receptors 5.2.4. Presynaptic actions of glutamate 5.2.5. Inactivation and uptake of glutamate

Diversification of synaptic strength: presynaptic elements

Synapses are not static; their performance is modified adaptively in response to activity. Presynaptic mechanisms that affect the probability of transmitter release or the amount of transmitter that is released are important in synaptic diversification. Here, we address the diversity of presynaptic performance and its underlying mechanisms: how much of the variation can be accounted for by variation in synaptic morphology and how much by molecular differences? Significant progress has been made in defining presynaptic structural contributions to synaptic strength; by contrast, we know little about how presynaptic proteins produce normally observed functional differentiation, despite abundant information on presynaptic proteins and on the effects of their individual manipulation. Closing the gap between molecular and physiological synaptic diversification still represents a considerable challenge.

Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons.

Stimulation of the excitatory axon of the opener muscle of the crayfish in the presence of the metabolic inhibitor 2,4-dinitrophenol leads to depletion of synaptic vesicles in nerve terminals containing round vesicles. Stimulation of the inhibitory axon under these conditions produces depletion of vesicles in other nerve terminals containing more elongate synaptic vesicles. The experiments show that terminals with round synaptic vesicles are excitatory and that terminals with elongate synaptic vesicles are inhibitory. Replenishment of synaptic vesicles appears to require metabolic energy.

Drosophila Hsc70-4 Is Critical for Neurotransmitter Exocytosis In Vivo

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus

Quantal content of transmission was estimated for three synaptic systems (crayfish and Drosophila neuromuscular junctions, and rat dentate gyrus neurons) with three different methods of measurement: direct counts of released quanta, amplitude measurements of evoked and spontaneous events, and charge measurements of evoked and spontaneous events. At the crayfish neuromuscular junction, comparison of the three methods showed that estimates from charge measurements were closer to estimates from direct counts, since amplitude measurements were more seriously affected by variable latency in evoked release of quantal units. Thus, charge measurements are better for estimating quantal content when direct counts cannot be made, as in crayfish at high frequency of stimulation or in the dentate gyrus neurons. At the Drosophila neuromuscular junction, there is almost no latency variation of quantal release in realistic physiological solutions, and the methods based upon amplitudes and charge give similar results. Distributions of evoked synaptic quantal events obtained by direct counts at the crayfish neuromuscular junction were compared to statistical distributions obtained by best fits. Binomial distributions with uniform or non-uniform probabilities of release generally provided good fits to the observations. From best fit distributions, the quantal parameters n (number of release sites) and p (their probability of release) can be calculated. We used two algorithms to estimate n and p: one allows for non-uniform probability of release and uses a modified chi-square (chi 2) criterion, and the second assumes uniform probability of release and derives parameters from maximum likelihood estimation (MLE). The bootstrap estimate of standard errors is used to determine the accuracy of n and p estimates.

Synaptic facilitation: long-term neuromuscular facilitation in crustaceans.

Continuous stimulation at frequencies equal to or greater than 5 hertz for 20 to 30 minutes results in a two- to fivefold increase in the amplitudes of excitatory postsynaptic potential recorded from certain stretcher and opener muscles of decapod crustaceans. This long-term facilitation appears to result from an accumulation of sodium ions within the nerve terminals. It persists for at least 1 hour after stimulation has stopped.

QUANTAL RELEASE AT VISUALIZED TERMINALS OF A CRAYFISH MOTOR AXON : INTRATERMINAL AND REGIONAL DIFFERENCES

Synaptic transmission was measured at visualized terminal varicosities of the motor axon providing the sole excitatory innervation of the “opener” muscle in walking legs of crayfish (Procambarus clarkii Girard). Two questions were addressed: 1) How uniform is quantal emission at different locations along terminals innervating a single muscle fiber, and 2) can differences in quantal emission account for the different excitatory postsynaptic potential (EPSP) amplitudes generated by terminals localized in defined regions of the muscle?

Presynaptic Ryanodine Receptor-Activated Calmodulin Kinase II Increases Vesicle Mobility and Potentiates Neuropeptide Release

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.